Directory UMM :Data Elmu:jurnal:J-a:Journal of Experimental Marine Biology and Ecology:Vol250.Issue1-2.Jul2000:

250 (2000) 133–167

www.elsevier.nl / locate / jembe

Review of nitrogen and phosphorus metabolism in seagrasses

*

Brant W. Touchette , JoAnn M. Burkholder

Department of Botany, Box 7510, North Carolina State University, Raleigh, NC 27695-7510, USA

Abstract

Within the past few decades, major losses of seagrass habitats in coastal waters impacted by cultural eutrophication have been documented worldwide. In confronting a pressing need to improve the management and protection of seagrass meadows, surprisingly little is known about the basic nutritional physiology of these critical habitat species, or the physiological mechanisms that control their responses to N and P gradients. The limited available evidence to date already has revealed, for some seagrass species such as the north temperate dominant Zostera marina, unusual responses to nutrient enrichment in comparison to other vascular plants. Seagrasses derive N and P from sediment pore water (especially ammonium) and the water column (most nitrate). The importance of leaves versus roots in nutrient acquisition depends, in part, on the enrichment conditions. For example, a shift from reliance on sediment pore water to increased reliance on the overlying water for N and P supplies has been observed under progressive water-column nutrient enrichment. Seagrasses may be N-limited in nutrient-poor waters with sandy or (less so) organic sediments, and P-limited in carbonate sediments. On the basis of data from few species, seagrasses

2 23 1

appear to have active uptake systems for NO3 and PO4 , but NH4 uptake may involve both low-and high-affinity systems. P uptake affinities reported thus far are much lower than values for_i active ammonium uptake, but comparable to values for nitrate uptake by leaf tissues. Beyond such basic information, seagrass species have shown considerable variation in nutritional response. Dominance of acropetal versus basipetal nutrient translocation appears to vary among species as an innate trait. While some species follow classic Michaelis–Menten kinetics for N uptake, others_i have exhibited sustained linear uptake with limited or negligible product feedback inhibition, perhaps in adaptation to oligotrophic environments. Zostera marina also is able to maintain nitrate reductase (NR) activity during dark periods if adequate carbohydrate reserves and substrate are available. Thus, this species can respond to nitrate pulses throughout a diel cycle, rather than being limited as most plants to nitrate uptake during the light period. Further adaptations may have occurred for seagrasses in extremely nitrate-depauperate conditions. For example, Halophila decipiens and H. stipulacea lack inducible NR and apparently have lost the ability to reduce nitrate; and a biphasic rather than hyperbolic P uptake curve, with ‘surge’ uptake, has been_i described for Zostera noltii. Many seagrasses respond favorably to low or moderate N and / or P enrichment. However, excessive N loading to the water column can inhibit seagrass growth and_i survival, not only as an indirect effect by stimulating algal overgrowth and associated light

*Corresponding author.

0022-0981 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 2 2 - 0 9 8 1 ( 0 0 ) 0 0 1 9 5 - 7

reduction, but—for some species—as a direct physiological effect. The latter direct impact has been most pronounced for plants growing in sandy (nutrient-poor) sediments, and is exacerbated by elevated temperatures and / or light reduction. Ammonia toxicity, known for many vascular plants, has been reported in seagrasses Ruppia drepanensis and Z. marina (125mM water-column

1

NH , 5 weeks). Z. marina has shown to be inhibited, as well, by pulsed water-column nitrate4

2

enrichment (as low as 3.5–7mM NO , 3–5 weeks), which is actively taken up without apparent₃ product feedback inhibition. Inhibition by elevated nitrate has also been reported, with description of the underlying physiological mechanisms, in certain macroalgae and microalgae. In Z. marina, this effect has been related to the high, sustained energy demands of nitrate uptake, and to inducement of internal carbon limitation by the concomitant ‘carbon drain’ into amino acid assimilation. In contrast, nitrate enrichment can stimulate growth of Z. marina when the sediment, rather than the water column, is the source. Because seagrass species have shown considerable variation in nutritional response, inferences about one well-studied species, from one geographic location, should not be applied a priori to that species in other regions or to seagrasses in general. Most of the available information has been obtained from study of a few species, and the basic nutritional physiology of many seagrasses remains to be examined and compared across geographic regions. Nonetheless, the relatively recent gains in general understanding about the physiological responses of some seagrass species to nutrient gradients already have proven valuable in both basic and applied research. For example, physiological variables such as tissue C:N:P content have begun to be developed as integrative indicators of nutrient conditions and anthropogenic nutrient enrichment. To strengthen insights for management strategies to optimize seagrass survival in coastal waters adjacent to exponential human population growth and associated nutrient inputs, additional emphasis is critically needed to assess the role of variable interactions—among inorganic as well as organic N, P and C, environmental factors such as temperature, light, and other community components—in controlling the physiology, growth and survival of these ecologically important marine angiosperms.  2000 Elsevier Science B.V. All rights reserved.

Keywords: Carbon; Light; Nitrogen; Phosphorus; Seagrasses; Temperature

1. Introduction

Seagrasses are highly productive marine angiosperms that grow in shallow coastal marine waters, often in sheltered embayments and lagoons which are poorly flushed and sensitive to nutrient loading from adjacent human population growth (Harlin, 1993). Seagrasses provide critical habitat and a nutritional base for finfish, shellfish, waterfowl,

˜

and herbivorous mammals (Phillips and Menez, 1988; Klumpp et al., 1989). In the past few decades, major declines in seagrass meadows have been reported worldwide, attributed to light reduction from land disturbance and sediment loading / resuspension, and light reduction from algal overgrowth that is stimulated by cultural eutrophication (Harlin, 1993; Morris and Tomasko, 1993). Under accelerated eutrophication, the severity of sediment hypoxia / anoxia and sulfide reduction that are encountered by seagrass root-rhizome tissues increase, as well (Mackin and Swider, 1989; Dauer et al., 1993; Goodman et al., 1995).

˜

of most seagrass species (McRoy, 1974; Phillips and Menez, 1988). However, this concept has become dogma, and should not be applied a priori to seagrass systems (Burkholder et al., 1992). Sediment pore waters can provide high supplies of most nutrients for seagrasses (except carbon), with the water column as an additional source (McRoy et al., 1972; Short and McRoy, 1984; Harlin, 1993). Thus, nutrients are commonly regarded as secondary factors limiting growth. The relative importance of nutrients as limiting factors increases for plants growing in sandy, nutrient-poor substrata and nutrient-poor waters. As a generalization, seagrasses are regarded as N-limited when growing in sandy or organic sediments, and as P-limited in carbonate sediments (Short, 1987; Short et al., 1990; Williams, 1990; Kenworthy and Fonseca, 1992; but see Zimmerman et al., 1987). In certain conditions, seagrasses may be co-limited by N and P (Thursby, 1984; Udy and Dennison, 1997a).

Although the influence of light availability on seagrass growth has been examined at length (e.g., Dennison et al., 1993), much less emphasis has been directed toward understanding the nutritional ecology of seagrasses, or their physiological responses to nitrogen and phosphorus enrichments (Burkholder et al., 1992). A scan of the literature in 1990, for example, yielded few published studies on aspects of nitrogen uptake and metabolism by Zostera marina, the dominant seagrass species in temperate and arctic regions of North America. Within the past decade, however, nutritional ecology and physiology have become more of a central focus in seagrass research. Here we synthesize the available information known about nitrogen and phosphorus metabolism of seagrasses, and the interplay between N, P, carbon, and environmental factors such as temperature and light in controlling the growth and survival of these ecologically important marine angiosperms.

2. N and P regimes in seagrass ecosystems

Reports of N limitation in seagrasses often seem counter-intuitive, since sediment

2

inorganic nitrogen availability (N , as NOi 3 and as ammonia which is mostly ionized as

1

NH ) would appear to be sufficient to sustain active, unlimited growth (Zimmerman et4

al., 1987). Nonetheless, many studies with N fertilization have demonstrated increased seagrass growth with increased N (Table 1). One possible explanation for N-limitedi

growth in natural systems is competition between seagrasses and other community flora such as micro- and macroalgae (Harlin, 1993), as well as microheterotrophs which can assimilate high proportions of regenerated ammonium (40% or more; Iizumi et al., 1982; Thayer et al., 1984). Ammonium also adsorbs to sediment and detrital particles (Simon, 1989). Thus, although ammonium levels may appear to be relatively high, the actual ammonium available for seagrasses may be substantially lower (Thayer et al., 1984).

Nitrate is the primary form of N available to terrestrial plants (Wu et al., 1998;i

Jenkins et al., 1999). However, in the water-saturated, hypoxic or anoxic sediments that characterize seagrass habitats, ammonium is considered to be the dominant form of Ni

(Riley and Chester, 1989; Stapel et al., 1996; Stumm and Morgan, 1996). Levels range

1

from 1 to 180 mM NH4 in sediment pore water, depending on sediment characteristics (especially percent organic matter) and community composition (Bulthuis and

Woekerl-Table 1

a

Overview of seagrass responses to nutrient enrichment and / or eutrophication events

Species Response Mechanism Nutrient Source

Temperate

Heterozostera tasmanica Increased growth N limitation N & P Bulthuis and Woekerling (1981)

Heterozostera tasmanica Increased growth N limitation N Bulthuis et al. (1992)

Posidonia oceanica Increased growth N & P limitation N & P Alcoverro et al. (1997) ´

Ruppia drepanensis Die-off Physiological N Santamarıa et al. (1994)

Ruppia maritima Increased growth N limitation N Burkholder et al. (1994)

Zostera capricorni Die-off Light attenuation N Abal and Dennison (1996)

Zostera capricorni Increased growth N & P co-limitation N & P Udy and Dennison (1997a,b)

Zostera marina Increased growth N limitation N Short (1987)

Zostera marina Increased growth N limitation N & P Kenworthy and Fonseca (1992)

Zostera marina Increased growth N limitation N Pedersen and Borum (1993)

Zostera marina Die-off Physiological N Burkholder et al. (1992, 1994)

Zostera marina Die-off Epiphytes N & P Neckles et al. (1993)

Zostera marina Die-off Macroalgal growth N Boynton et al. (1996)

Zostera marina Die-off Light attenuation N Lent et al. (1995)

Zostera marina Die-off Light attenuation N & P de Casabianca et al. (1997)

Zostera noltii Die-off Light attenuation N & P de Casabianca et al. (1997)

Tropical / subtropical

´

Cymodocea nodosa Increased growth P limitation P Perez et al. (1994)

Cymodocea serrulats No effect N & P Udy and Dennison (1997a,b)

Enhalus acoroides Increased growth N limitation N & P Agawin et al. (1996)

Halodule uninervis Increased growth N limitation N & P Udy and Dennison (1997a,b)

Halodule wightii Increased growth N & P limitation N & P Powell et al. (1989) ´

Cymodocea nodosa Increased growth P limitation P Perez et al. (1994)

Cymodocea serrulats No effect N & P Udy and Dennison (1997a,b)

Enhalus acoroides Increased growth N limitation N & P Agawin et al. (1996)

Halodule uninervis Increased growth N limitation N & P Uddy and Dennison (1997a,b)

Halodule wrightii No effect N & P Kenworthy and Fonseca (1992)

Halodule wrightii Increased growth N limitation N Burkholder et al. (1994)

Syringodium filiforme Increased growth N limitation N Short et al. (1993)

Syringodium filiforme Increased growth P limitation P Short et al. (1990)

Thalassia hemprichii Increased growth P limitation N & P Agawin et al. (1996)

Thalassia testudinum Die-off Microbial associations N Durako and Moffler (1987)

Thalassia testudinum Increased growth N and P limitation N & P Powell et al. (1989)

Thalassia testudinum Die-off Macroalgal growth N & P McGlathery (1995)

Thalassia testudinum Die-off Light attenuation N Tomasko et al. (1996)

a

Data include seagrass species (temperate and tropical / subtropical), growth response, possible mechanisms for the observed responses, nutrients involved, and source.

ing, 1981; Fourqurean et al., 1992a; Lee and Dunton, 1999; Table 2). Total water-column Ni is typically less than 3 mM during light periods in habitats lacking appreciable land-based anthropogenic influence as freshwater inflow (Table 2; Tomasko

1

and Lapointe, 1991; Dunton, 1996; but see Lee and Dunton, 1999). Higher NH4 in both the water column and sediment pore water is available (as regenerated N ) at night on ai

diel basis, especially in warm seasons, because of increased decomposition from ecosystem metabolism and temporary low-oxygen conditions at the sediment–water

Table 2

Summary of water column and sediment nutrient concentrations observed in seagrass habitats, including

a

species, ammonium, nitrate1nitrite, and phosphate (all nutrients inmM), location, and source

1 2 2 23

Species NH4 NO31NO2 PO4 Location Source

Water column

Temperate

Phyllospadix torreyi 0.42 0.40 – S. California, USA Terrados and Williams (1997)

Posidonia australis 0.47–0.5 0.08–0.1 0.13–0.25 Cockburn Sound, Australia Silberstein et al. (1986)

Zostera capricorni 0.7 ,0.1 0.53 Moreton Bay, Australia Boon (1986)

Zostera marina up to 8 1.0 Chesapeake Bay, USA Moore et al. (1996)

Tropical / subtropical

Cymodocea serrulata 0.7 ,0.1 0.53 Moreton Bay, Australia Boon (1986)

Enhalus acoroides 1.4 0.6 1.7 Indonesia Erftemeijer (1994)

Enhalus acoroides 1.4 0.9 0.8 Indonesia Erftemeijer (1994)

Thalassia hemprichii 1.4 0.9 0.8 Indonesia Erftemeijer (1994)

Thalassia hemprichii 1–2.6 – 0.1–0.6 Indonesia Stapel et al. (1996)

Thalassiaa testudinum 0–3.2 0.05–2.3 0–0.43 Florida Keys, USA Tomasko and Lapointe (1991)

Sediment

Temperate

Phyllospadix torreyi 9.6 4.8 – S. California, USA Terrados and Williams (1997)

Zostera capricorni 6.7–11.5 – 0.7–6.3 Moreton Bay, Australia Udy and Dennison (1997a,b)

Zostera capricorni 29–59 3–10 3–17 Moreton Bay, Australia Boon (1986)

Tropical / subtropical

Cymodocea serrulata 37–57 5–6 6–20 Moreton Bay, Australia Boon (1986)

Enhalus acoroides 0.8 2.2 0.9 Indonesia Erftemeijer and Middelburg (1995)

Enhalus acoroides 47–90 – 3.5–7 Indonesia Erftemeijer and Middelburg (1993)

Halodule wrightii 10–50 – – Caribbean Williams (1990)

Halodule wrightii 150–175 – 2–4 Florida Bay, USA Fourqurean et al. (1992a,b)

Syringodium filiforme 10–50 – – Caribbean Williams (1990)

Thalassia hemprichii 60–136 – 0.92–5.5 Indonesia Stapel et al. (1996)

Thalassia hemprichii 0.8 2.2 0.9 Indonesia Erftemeijer and Middelburg (1995)

Thalassia hemprichii 25–60 – 6–16 Indonesia Erftemeijer and Middelburg (1993)

Thalassia testudinum 8–180 – – S. Texas, USA Lee and Dunton (1999)

Thalassia testudinum 60–120 – 0.3–0.8 Florida Bay, USA Fourqurean et al. (1992a,b)

Thalassia testudinum 10–50 – – Caribbean Williams (1990)

a

Data are given as means, or as ranges if means were not available.

interface (Henriksen and Kemp, 1988). A considerable portion of the available Ni 1

(NH ) in seagrass beds can also be provided by cyanobacterial (blue–green algal) and4

eubacterial nitrogen fixation (Capone et al., 1979).

Nitrate supplies for most seagrasses are provided by leaf absorption from the water column (Terrados and Williams, 1997; Lee and Dunton, 1999), except for groundwater

2

inflows that can contribute as much as 800mM NO N to seagrass beds if influenced by3

septic effluent leachate or other anthropogenic sources (Maier and Pregnall, 1990; Harman et al., 1996). Leakage of oxygen from the lacunar system can create a microaerobic environment immediately adjacent to roots (Penhale and Wetzel, 1983), which can oxidize ammonia to nitrate as a mechanism for belowground tissue uptake of this highly soluble N form (Iizumi et al., 1980; Stumm and Morgan, 1996). However,i

most nitrate is regarded as ‘new’ nitrogen for the system, contributed from anthro-pogenic rather than regenerated inputs (Glibert, 1988; Henriksen and Kemp, 1988). Aside from N sources, seagrasses can also utilize certain organic N substrates such asi

amino acids (product of decomposition) and urea (waste product from various fauna in seagrass communities; also contributed by poorly treated human and animal wastes; Vitousek et al., 1997; see below).

Phosphorus levels within seagrass habitats range from ca. 0.1 to 1.7mM (as available

23

dissolved phosphate, PO4 P) in the water column, with higher concentrations in the

23

sediment pore water (0.3–20mM PO4 P; Table 2). Most P absorption for seagrasses isi

believed to occur through the roots (McRoy et al., 1972; but see below). Phosphate is highly biologically active, with a characteristically short residence time in the water column (Day et al., 1989). Like N , it is consumed by seagrasses, other community florai

¨

such as micro- and macroalgae (Harlin, 1993), and microheterotrophs (Gachter and

23

Meyer, 1993; Slomp et al., 1993). And, like ammonium, higher levels of PO4 are available in both the water column and sediment pore water at night on a diel basis, especially in warm seasons, because of increased decomposition and sediment release under low-oxygen conditions (Day et al., 1989). P is also highly insoluble and readilyi

adsorbs to organic and inorganic particles (Froelich, 1988; Stumm and Morgan, 1996). In marine environments, especially in tropical and subtropical areas that are dominated by carbonate sediments, P can be effectively removed from access by seagrassesi

through formation of calcium phosphate complexes (e.g., Ca (PO ) ; Garrels et al.,3 4 2

1975; Stumm and Morgan, 1996).

3. Nitrogen physiology

The physiology for N assimilation to amino acids by plant tissues is complex,i

especially when the N form is nitrate, and involves various biochemical pathways thati

occur within the cytosol, the chloroplast, and the mitochondria (Fig. 1). Whereas nitrate is taken up via an active transport system (see below), ammonium uptake may be more complex. The electrochemical gradient between the apoplast and the symplast of plants traditionally has been viewed as a region of passive movement / uptake of small cations,

1

including NH , through membrane channels (Ourry et al., 1997). Recently, however,4

studies on agriculturally important plants have revealed active enzyme-like uptake

1 1

responses to NH , including substrate saturation at low NH4 4 levels (e.g., Wang et al., ¨

1993; Mack and Tischner, 1994). Moreover, plants grown in low-N or N-free media

1 1

have been shown initially to have low NH4 uptake rates after NH4 additions, followed by an induction-like response with increased uptake rates after several hours (Ourry et

1

al., 1997). These data indicate that NH uptake may involve two separate transport4 1

systems—a low-affinity system based on passive influx and efflux of NH4 through membrane channels, and a high-affinity system requiring protein synthesis for formation of uniports (transmembrane transport protein that conveys a single species across the plasma membrane; Ourry et al., 1997). It is also possible, given the product feedback characteristic of many high-affinity systems (Goodwin and Mercer, 1983), that the

Fig. 1. Theoretical nutrient uptake curve, with parameters based on the Michaelis–Menton model for uptake kinetics (adapted from Cornish-Bowden, 1979). Parameters include Vmax(maximum uptake rate), Km (half-saturation constant51 / 2Vmax),a(uptake affinity5Vmax/K ), and Sm min(minimum substrate level for uptake to occur).

1

assimilated NH4 may exert a negative feedback on further uptake when tissue ammonia levels are high (Lee and Ayling, 1993).

Although most available information about N acquisition by seagrasses has empha-sized N forms, a few seagrass species have been shown to be capable of organic Ni

uptake as amino acids and urea (McRoy and Goering, 1974; Bird et al., 1998). Surprisingly, up to 20% of the total N supply in vascular plants has been reported to be derived from amino acids (Schobert and Komor, 1987; Bush, 1993). Amino acid uptake

1

apparently requires H ATPase pumps that promote membrane depolarization as well as alkalinization of the external medium (Ourry et al., 1997). Information on urea assimilation by seagrasses is extremely limited: McRoy and Goering (1974) showed urea uptake by field-collected Zostera marina, whereas Bird et al. (1998) reported that

Halophila decipiens grown in tissue culture did not utilize urea.

3.1. N acquisition by above- and belowground tissues

Young, actively growing roots have been reported to take up most of the N absorbed by belowground tissues in seagrasses, with rhizomes functioning minimally in N uptakei

(Short and McRoy, 1984; Barnabas, 1991; Stapel et al., 1996). Although sediment pore water is generally considered to be the primary N source for seagrasses, recent evidencei

suggests that uptake of both N and P by belowground tissues can be limited by diffusion, and that roots may lack the capacity to support the total nutrient requirement (Stapel et al., 1996; Lee and Dunton, 1999). Thus, experiments by Lee and Dunton (1999)

indicated that about 50% of the nitrogen uptake in Thalassia testudinum occurs through the leaves. Other studies suggest that from 30 to 90% of the N requirement in Zostera

marina can be supplied by the water column (Iizumi and Hattori, 1982; Short and

McRoy, 1984; Pedersen and Borum, 1992, 1993). In extreme cases—for example, on rock substrata (as for Phyllospadix torreyi and, to some extent, Amphibolis antarctica)— nearly all nutrient absorption occurs in the leaves (Terrados and Williams, 1997).

The role of leaves in nutrient acquisition may partly be explained by the relatively high nutrient uptake affinities of these tissues. Many seagrasses can assimilate Ni

through their leaves at extremely low levels (Pedersen et al., 1997; Lee and Dunton, 1999). This response may reflect an adaptation of seagrasses to oligotrophic environ-ments (Burkholder et al., 1994). It allows plants to acquire pulsed low-level N as iti

enters the system, while also absorbing nutrients that diffuse from the sediments, or that are released from detrital decomposition (Paling and McComb, 1994; Stapel et al., 1996).

In Zostera marina, N (as N and urea) assimilated by roots can be moved toi

aboveground tissues (acropetal or root-to-shoot translocation; McRoy and Goering, 1974). Conversely, assimilated N from leaves can be translocated to belowgroundi

tissues (basipetal or shoot-to-root translocation; Iizumi and Hattori, 1982). Ammonium

1

uptake by aboveground tissues is reportedly unaffected by NH4 concentrations available to the roots, even at sediment pore water concentrations in excess of 150mM (Thursby and Harlin, 1982). In contrast, maximum rates of root ammonium uptake were

21 21

substantially compromised (decreased from 211 to 48mmol g dry weight h ) during periods when leaves were exposed to increased ammonia (15–30 mM; Thursby and Harlin, 1982). It was hypothesized that the observed response was associated with higher basipetal translocation of N products relative to acropetal translocation.

These characteristics of N uptake by Z. marina tissues may explain, in part, the sustained uptake of water-column nitrate enrichment by Z. marina that has been observed in other studies (e.g., Roth and Pregnall, 1988; Burkholder et al., 1994— indicated by nitrate reductase activity; see below), even when sediment N (as ammonia)i

appears to be at levels required for optimal growth (Thursby and Harlin, 1982; but see Roth and Pregnall, 1988, below). In contrast to the somewhat independent response of Z.

marina leaves to belowground tissue N availability, uptake of ammonium by Ruppiai

maritima leaves has been described to be strongly affected by ammonium supplies to the

roots (Thursby and Harlin, 1984). Acropetal translocation may be dominant in this plant, or roots may be unaffected by internal whole-plant ammonium status (Thursby and Harlin, 1984).

3.2. Nitrogen uptake kinetics

For seagrass species that follow Michaelis–Menten kinetics in N absorption, thei

uptake affinity coefficient (a, defined as the initial slope of the V versus S curve; Table 3, Fig. 2) is useful in evaluating the efficiency of nutrient uptake within the concentration range characteristic of natural conditions (e.g., Stapel et al., 1996). In leaf tissues, uptake

1

affinities for N are highly variable, ranging from 0.5 to 5.5 for NHi 4 and from 0.15 to

2 1 2

Table 3

Nutrient uptake parameters reported in seagrass species based on the Michaelis–Menten model for uptake

21 21

kinetics, including tissue (leaf, root), nutrient, Vmax (maximum uptake rate,mmol g dry weight h ), Km

(half-saturation constant,mM), anda (uptake affinity5Vmax/K )m

Species Nutrient Vmax Km a Source

Leaf

Temperate

1

Amphibolis antarctica NH4 5.9–43.1 9.5–74.3 0.6–0.8 Pedersen et al. (1997)

1

Phyllospadix torreyi NH4 95.6–204.3 9.3–33.9 – Terrados and Williams (1997)

2

Phyllospadix torreyi NO3 24.9–75.4 4.4–17.0 – Terrados and Williams (1997)

1

Ruppia maritima NH4 243–270 9.0–17.7 5.5 Thursby and Harlin (1984)

1

Zostera marina NH4 20.5 9.2 2.2 Thursby and Harlin (1982)

–

Zostera marina NO3 – 23 – Iizumi and Hattori (1982)

a – 3

´ ´

Zostera noltii (excised) PO4 7.0 10 0.7 Perez-Llorens and Niell (1995)

a 23

´ ´

Zostera noltii PO4 43 12.1 1.1 Perez-Llorens and Niell (1995)

Tropical / subtropical

a 23

Thalassia hemprichii PO4 2.2–3.2 7.7–15 0.12–0.19 Stapel et al. (1996)

1

Thalassia hemprichii NH4 32–37 21–60 0.52–0.85 Stapel et al. (1996)

1

Thalassia testudinum NH4 8.3–16.4 7.6–15 0.57–2.82 Lee and Dunton (1999)

2

Thalassia testudinum NO3 3.7–6.5 2.2–38.5 0.15–1.68 Lee and Dunton (1999)

Root

Temperate

1

Amphibolis antarctica NH4 1.1 4.7 0.2 Pedersen et al. (1997)

1

Ruppia maritima NH4 48–56 2.8–12.6 20.1 Thursby and Harlin (1984)

1

Zostera marina NH4 211 104 0.5 Thursby and Harlin (1982)

1

Zostera marina NH4 – 30 – Iizumi and Hattori (1982)

Tropical / subtropical

1

Thalassia testudinum NH4 7.9–73.3 34.4–765.5 0.03–0.30 Lee and Dunton (1999)

a

Note: minimum substrate level (Smin) was also available for Zostera noltii (excised leaf, 2.5mM; leaf, 2.6

mM) and Thalassia hemprichii (leaf, 0.7–1.1mM).

many seagrass species (e.g., Amphibolis antarctica, Thalassia testudinum, Zostera

1

marina), calculated uptake affinities for NH4 are higher in leaves than in roots (Table 3; root values highly variable, ranging from 0.03 to .20). Thus, seagrass leaves tend to be more efficient than roots in absorbing low levels of ammonium—a response that likely

1

reflects the low levels of NH4 in the water column relative to the sediments (Lee and Dunton, 1999).

1

Seagrass leaves generally have higher uptake rates for for NH4 (between 5 and 270

21 21 2 21 21

mmol g dry weight h ) than for NO3 (3.7 to 75mmol g dry weight h ). This trend has been shown for Amphibolis antarctica, Phyllospadix torreyi, Thalassia

testudinum and Zostera marina (Short and McRoy, 1984; Paling and McComb, 1994;

Terrados and Williams, 1997; Lee and Dunton, 1999). Although these rates vary seasonally, Lee and Dunton (1999) reported negligible variations in Vmaxand K in lightm

1 2

versus dark assimilation of NH4 (roots and leaves) or NO3 (leaves) by T. testudinum. Nitrate uptake by Z. marina in response to pulsed inputs has been experimentally shown in both darkness and light, as well (Touchette, 1999; see below). Thus, some seagrasses can develop the capability to absorb N regardless of light conditions. This characteristici

Fig. 2. Reactions involved in nitrate uptake and assimilation in plant cells. Uptake and assimilation of nitrate as well as ammonium occur primarily in the cytosol and plastids (chloroplasts, mitochondria). As nitrate enters the cell, it can be stored in a vacuole (not shown) or reduced in the cytosol by the enzyme nitrate reductase (NR). The product, nitrite, enters the plastids (usually the chloroplasts) where it is further reduced to ammonium by the enzyme nitrite reductase (NiR). Ammonium then enters the GS / GOGAT cycle, where glutamate is aminated by the enzyme glutamine synthetase (GS) to synthesize glutamine. The addition of carbon skeletons (a-ketoglutarate) allows transamination by glutamate synthase (GOGAT) to produce two glutamate molecules. One of these can be recycled through the GS / GOGAT pathway, whereas the other can

´

be used to form more complex amino acids (adapted from Ferrario-Mery et al., 1997).

would be especially advantageous for seagrasses in oligotrophic habitats, by enabling them to utilize N supplies from nocturnal storm events or other source of nutrienti

pulses.

The capacity for nutrient uptake (Vmax, which can be considered as an indication of the number of active uptake sites; Thursby and Harlin, 1984) also varies in response to changing nutrient conditions. In Ruppia maritima, for example, Vmax was significantly lower in N-starved than in N-replete plants (Thursby and Harlin, 1984). This observation may have indicated the presence of a feedback inhibition mechanism for plants growing in eutrophic conditions. In Zostera marina leaves, a similar adjustment in Vmax was

1

reported following long-term exposure (e.g., 24 h) to increased NH4 (Short and McRoy, 1984). The latter research involved time-series incubations on plants in situ at a location

1

that was described as N-limited; thus, the biphasic NH4 uptake response may have been symptomatic of initial N starvation, perhaps followed by product feedback inhibition.

Although some seagrasses have followed Michaelis–Menten kinetics for N uptake,i

other species have shown more variable response; or, the same seagrasses have not followed Michaelis–Menten uptake kinetics under other conditions. For example, seedlings of the seagrass Amphibolis antarctica showed a direct linear relationship

2 1

between uptake rates and N concentrations (from 0.3 to 36.0i mM of NO3 and NH )4

and, thus, a Michaelis–Menten analytical approach was not applicable (Paling and McComb, 1994). The seedlings survived and grew as leaf clusters with little or no root development, so that N uptake was restricted mostly to leaves until the plants hadi

matured. Given this inability to directly utilize sediment N , it is reasonable that A.i

antarctica seedlings would maintain linear uptake of N at high water-column levelsi

when such pulses became available.

Studies of N assimilation in several seagrass species have shown limited or negligiblei 1

product feedback inhibition. For example, NH4 uptake kinetics in the seagrass Thalassia

hemprichii indicated no evidence of a feedback inhibition mechanism (Stapel et al.,

1996). The authors of that study hypothesized that the lack of this mechanism was indicative of an adaptation to oligotrophic environments. This lack of an apparent substrate saturation or product feedback inhibition may provide insights about adaptive responses of seagrasses to changing nutrient regimes. Research by Roth and Pregnall (1988) illustrates this point: Zostera marina was collected from sandy (low N) versus organic (high N) sediments and low-N overlying waters. Nitrate uptake was compared

2

under N-enriched conditions (laboratory trials, 6 days, 200mM NO ). The plants grown3

in sandy sediments showed a linear response in nitrate uptake, with no evidence of product feedback inhibition over time. A linear response in nitrate uptake was also shown by the plants from N-rich sediments, but uptake was significantly lower. The data indicate that the low N-adapted plants had conformed to maximize their response to

2

water-column NO3 enrichment, relative to the plants that had been growing in sediment N-enriched conditions.

3.3. Ammonium assimilation

The glutamine synthetase (GS) / glutamate synthase (glutamine-oxoglutarate amido-transferase or GOGAT) pathway of ammonium incorporation (Goodwin and Mercer, 1983) is the primary biochemical cycle involved in ammonium assimilation. This pathway involves initial incorporation of ammonium to glutamate by GS, using ATP to catalyze formation of glutamine (localized in the cytosol and chloroplast of photo-synthetic tissues, and in the cytosol of roots; Emes and Fowler, 1979; Oaks and Hirel, 1985). Glutamate may then be regenerated from glutamine and a-ketoglutarate by the enzyme GOGAT (localized in the chloroplast of photosynthetic tissues, and in root plastids; Oaks and Hirel, 1985), for use in production of new amino acids or in completing the GS / GOGAT pathway as the initial substrate for ammonium assimilation. The GS / GOGAT system is also involved in secondary assimilation of ammonium that can be released from glycine during photorespiration—a mechanism that is regarded as essential for N conservation within plants (Goodwin and Mercer, 1983).

Ammonium, partly because of its toxicity as a decouplet of photophosphorylation in chloroplasts (Marschner, 1995), does not accumulate in healthy seagrass tissues, thus

indicating rapid assimilation into organic compounds (Touchette, 1999). Most of the research on ammonium assimilation in higher plants has focused on GS activity because it is believed to be the key enzyme involved in synthesis of amino acids (Temple and Sengupta-Gopalan, 1997). GS activity of both belowground (e.g., Zostera marina; Kraemer and Alberte, 1993) and aboveground tissues (e.g., Posidonia oceanica; Kraemer et al., 1997) decreases with tissue age. Moreover, GS activity is higher in above- than in belowground tissues (e.g., 50-fold higher in leaves than in roots; Kraemer et al., 1997). These data, like the data for N uptake kinetics, indicate the importance of aboveground tissues in N assimilation.

GS activity in seagrasses is highly influenced by the surrounding environment. For example, Kraemer et al. (1997) demonstrated that leaf GS activity in P. oceanica varies with depth, season, and location. Highest GS activity was observed during active growth periods as expected, and for plants in deeper waters (for both Z. marina and P. oceanica; Pregnall et al., 1987; Kraemer et al., 1997). Moreover, as in terrestrial plants (e.g., Kozaki et al., 1991; Seith et al., 1994; Sukanya et al., 1994), increasing external Ni

supplies can promote significant increases in GS gene expression and activity in seagrasses (for Z. marina and P. oceanica; Pregnall et al., 1987; Kraemer et al., 1997). Kraemer et al. (1997) suggested that in vivo GS activity in P. oceanica may be limited

1

by the availability of the substrate (NH ), carbon skeletons (e.g., glutamate or4 a -ketoglutarate), and / or energy.

Temperature, hypoxia, and light availability (i.e., intensity and H , the duration ofsat

the daily light period when light equals or exceeds the photosynthetic light saturation point, I ) can also influence GS activity in seagrasses. GS activity in belowgroundk

tissues showed little response to N enrichment at temperatures above or below valuesi

known for optimal growth of Z. marina (i.e., 258C; Wetzel, 1982; Touchette, 1999). However, at ca. 258C, GS activity in belowground tissues was elevated during water-column nitrate enrichment (Touchette, 1999; Fig. 3). Furthermore, removal of reduced sediments had little effect on GS activity in Z. marina root tissues, whereas rates of ammonium assimilation were depressed during periods of root hypoxia / anoxia (Pregnall et al., 1987). Thus, internal root anaerobiosis and anoxia would appear to be more important in controlling GS activity than external rhizosphere redox conditions. Pregnall et al. (1987) suggested that increased GS activity was associated with shorter Hsat and longer periods of root anoxia for Z. marina in deeper waters. Plants growing in such habitats may increase GS activity to attain comparable ammonium assimilation rates as plants growing in shallow (less light-limited, less anoxic) habitats.

3.4. Nitrate assimilation

The assimilation of nitrate is an energy-requiring process that is highly regulated in plants, involving activation / production of nitrate-induced proteins specifically for nitrate active transport systems (Taiz and Zeiger, 1991; Larsson, 1994). Following increased exposure to nitrate, an inducible transport system is established wherein nitrate acts as the signal to stimulate uptake. This nitrate-specific transport system is inhibited by cyanide, anaerobic conditions, and an oxidative phosphorylation uncoupler

(dinitro-Fig. 3. Model of the typical response of the enzyme glutamine synthetase (GS) to high versus low inorganic nitrogen N availability in Zostera marina. During optimal growth temperatures, GS activity increases withi

increasing N . The optimum temperature for Z. marina was considered as 25i 8C (indicated by Wetzel [1982] for

Z. marina in southern Chesapeake Bay, and by Touchette [1999] for Z. marina in North Carolina coastal waters, at the southernmost extension of the range for this species on the US Atlantic Coast). As temperatures diverge in either direction from the 258C optimum, GS response to elevated N decreases substantially (basedi

on seasonal observations of Z. marina from Touchette, [1999]).

phenol), thus illustrating the importance of metabolic energy for its function (Taiz and Zeiger, 1991).

Although nitrate reduction can occur in roots, nitrate generally is translocated to leaves for storage in vacuoles and subsequent assimilation (known for terrestrial plants;

2 21

e.g., spinach can store nitrate in excess of 4.2 mg NO3 g fresh weight , levels that would be toxic to many animals; Steingrover et al., 1982). Tissue nitrate content tends to be significantly higher in above- than in belowground structures (e.g., nearly 2-fold higher in aboveground tissues of Z. marina; Touchette, 1999). However, in that species (perhaps indicative of N limitation), little nitrate storage apparently occurs even under

21

water-column nitrate enrichment (0–6.8mg g fresh weight in Z. marina under pulsed

2

enrichment at 8 mM NO3 for 14 weeks; Touchette, 1999).

Nitrate reductase (NR) is the key enzyme involved in nitrate assimilation, and the key ´

regulatory enzyme in nitrate metabolism (Ferrario-Mery et al., 1997). The presence of nitrate induces the de novo synthesis of NR which reduces nitrate to nitrite in the cytosol, using NADH (less commonly, NADPH) as an electron donor (Beevers and Hageman, 1983). The half-life of NR is only a few hours; thus, as environmental or cellular levels of nitrate decrease, NR activity rapidly declines (Hewitt et al., 1976). In many plants, NR activity tends to follow the light curve, with highest activity during photosynthetic periods and lowest activity in darkness (Layzell, 1990).

NR appears to be tightly regulated at multiple levels. For example, light / dark modulation of NR activity involves alterations in both transcription rates and protein

21

phosphorylation, which increases enzyme sensitivity to Mg inhibition (Huber et al., 1992). This mechanism, favoring light (photosynthetic) periods, allows plants to take advantage of periods when carbohydrates and reductants are most available, thereby providing the required reduction energy and carbon skeletons for nitrate assimilation (Kaiser, 1990; Turpin et al., 1991). Substrate concentration and light are considered to be the two major environmental factors that influence NR activity, but the activity of this enzyme is also affected by temperature, ammonium levels, dissolved oxygen, and carbon

¨

dioxide availability (Kenis and Trippi, 1986; de Cires et al., 1993; Muller and Janiesch, 1993). Plant physiological status (for example, carbohydrate content) is important in controlling NR response, as well (Kaiser, 1990; Berges et al., 1995).

Nitrite, the product of NR, is toxic to plant tissues (Ourry et al., 1997). To prevent accumulation, it must be further reduced to ammonium by nitrite reductase (NiR), a process that is catalyzed in plastids (typically, in chloroplasts). The reduction of nitrate to nitrite is energy-costly, requiring six electrons from the electron donor ferredoxin. The ferredoxin complex involved in nitrite reduction is the same complex used in the light reactions of photosynthesis (i.e., photosystem I; Goodwin and Mercer, 1983). Thus, there is competition for electrons between photosynthesis and nitrite reduction. Sea-grasses have negligible NR activity in belowground and sheath tissues, so that nitrite reduction probably occurs mostly in leaf tissues. The process involves a specific ferredoxin-NiR (Touchette, 1999; note that terrestrial angiosperms also have a small amount of NAD(P)H-dependent NiR in proplastids of non-green tissues; Ourry et al., 1997). The potentially toxic product of nitrite reduction, ammonium, cannot be allowed to accumulate as mentioned, and must be incorporated into amino acids through the GS / GOGAT pathway as previously described.

Although in vitro NR assays (using crude extracts of fresh or frozen tissues) are in general use, sources of unknown interference have caused significant underestimates of NR activity in seagrasses. Thus, in vivo techniques (involving incubations of living tissues) are recommended (Roth and Pregnall, 1988; Touchette, 1999). In vitro assays are more widely used in plant tissues because this approach limits the influence of substrate availability (e.g., electron donor NAD(P)H in Vmaxestimates) and, thus, enables assessment of maximum enzyme activity under optimal conditions. However, when considering the overall process of nitrate reduction, in vivo assays can provide valuable insights about the behavior of NR in situ (Abdin et al., 1992; Lee and Titus, 1992), including available energy for nitrate reduction and, thus, provides a valuable physiolog-ical approach.

Among the most intensively studied seagrasses for nitrate assimilation is the north temperate species, Zostera marina. NR in this seagrass is highly inducible and typically increases within 6 h following exposure to elevated water-column nitrate levels. Younger leaves have 2- to 5-fold higher activity than older leaves, and 10-fold higher activity than roots (Roth and Pregnall, 1988; Touchette, 1999). Environmental factors such as low P , hypoxia, and light deprivation can significantly depress in vivo NR activity in Z.i

marina (Touchette, 1999). As in terrestrial plants, light is important in NR regulation of Z. marina. NR activity is lower in plants growing at depth (where light availability is reduced) relative to that of plants from shallow waters, suggesting the importance of light on overall NR response (Roth and Pregnall, 1988).

Z. marina differs from many other plants in that some NR activity can be sustained throughout dark periods, provided that the surrounding water is nitrate-enriched (Gao et al., 1992; de Cires et al., 1993; Berges et al., 1995; Touchette, 1999; Fig. 4). However, the intensity and duration of NR activity is tightly coupled with, and directly related to, carbohydrate levels, wherein plants with relatively high soluble carbohydrate content tend to have much higher NR activity (Fig. 5). Therefore, if carbohydrates remain plentiful during dark periods, Z. marina can still assimilate and reduce nitrate at rates that are sometimes comparable to those measured in the light (Touchette, 1999). But during extended intervals of darkness or low light availability (for example, days of high turbidity following severe storm events), declines in available energy and carbohydrates coincide with a decline in NR activity (Touchette, 1999).

This apparent adaptation in Z. marina to assimilate water-column nitrate during dark periods (provided that adequate carbohydrate reserves are available) would provide a mechanism to enable response to pulsed nitrate enrichment whenever it occurred within a diel cycle. Nitrate is often added to coastal waters by terrestrial runoff which, in turn, is associated with storm events and increased turbidity (Kemp et al., 1983; Vitousek et al., 1997). N-Limited plants that could utilize available nitrate from the surrounding water during periods of darkness or low light would have a competitive advantage over plants that shut down nitrate assimilation in the dark. However, that not all seagrasses have inducible NR. For example, the subtropical seagrass Halophila stipulacea showed little inducible in vivo NR activity even following substrate additions as high as 1 mM

2

NO N for 42 h (Doddema and Howari, 1983). The low NR activity was not associated3

with ammonium suppression, as has been reported in other plants (Doddema et al., 1978; Doddema and Howari, 1983). It was hypothesized that H. stipulacea has adapted to extremely low-nitrate environments, and may have lost its ability to reduce nitrate.

3.5. Nitrogen inhibition

Udy and Dennison (1997a) proposed three categories of seagrass response to nutrient enrichment and the associated environmental conditions, paraphrased as follows: (i) growth and physiology respond favorably to the additions in low-nutrient habitats where nutrients are the only environmental factor limiting growth; (ii) there is a positive physiological response but no increase in growth, in low-nutrient habitats where environmental factors other than the added nutrient limits growth; and (iii) there is neither a growth nor a physiological response in high-nutrient environments where nutrient supplies are in excess. We propose an additional category of seagrass response, wherein (iv) there is a negative physiological response and an inhibition of growth by the added nutrient (e.g., Burkholder et al., 1992; van Katwijk et al., 1997).

It is well established that cultural eutrophication can stimulate algal overgrowth which reduces available light for seagrasses and promotes their decline (Borum, 1985; Twilley et al., 1985; Wear et al., 1999). However, recent seasonal mesocosm experiments, conducted at the southernmost extent of the range for Zostera marina, have documented significant lower survival of that species when grown in sandy sediments (1:3 mixture of sand to salt marsh organic muds) under water-column nitrate enrichment (pulsed daily

2

Fig. 4. Model comparing nitrate reductase (NR) activity in leaves of Zostera marina under nitrate enrichment to the generalized NR response that has been described for many plants (algae, terrestrial angiosperms, etc.; Goodwin and Mercer, 1983; Turpin, 1991) over a diel cycle (dark bars5night). (A) When nitrate is pulsed in the light period, NR activity significantly increases. During subsequent dark periods, NR activity decreases as a typical plant response. However, Z. marina can maintain NR activity in the dark, provided that nitrate is still available and carbohydrate supplies are abundant. (B) When nitrate is pulsed during the dark, NR activity in Z.

marina increases in response to the nitrate. In contrast, most plants do not respond to nitrate pulses in the dark,

and NR does not increase until the next light period (based on diel observations of Z. marina; Touchette, 1999).

Fig. 5. Relationship between in vivo nitrate reductase (NR) activity (mmol nitrite per gram dry weight per hour) and sucrose (mg per gram fresh weight) in Zostera marina leaf tissue (from whole-plant incubations), showing a comparison of control (un-enriched, white bars) and nitrate-enriched plants (black bars; pulsed

2

nitrate, 8mM NO ). Note that plants with relatively high sucrose levels had higher NR activity. Data are given3

as means61 S.E. (n$6; from Touchette, 1999).

documented in experimental series was unrelated to light reduction from algal over-growth or other source (Burkholder et al., 1992, 1994; Touchette, 1999). Treatments with water-column nitrate enrichment and light reduction in combination (from shading with neutral density screens), or with water-column nitrate enrichment and elevated temperature, led to significantly lower shoot survival (Touchette, 1999; Burkholder, 2000). This inhibitory physiological effect of nitrate enrichment was not demonstrated if the sediment, rather than the water column, was the substrate source, perhaps because of higher microbial reduction / utilization in sediments, a greater degree of diffusion-limited

1

uptake, lower energy requirements for NH4 assimilation (more abundant in sediments), and regulatory mechanisms that may be operable in the plant roots to limit nitrate absorption (Burkholder et al., 1992).

In the seasonal mesocosm experiments, the affected plants in water-column-enriched treatments showed no product feedback inhibition, that is, no ability to ‘shut off’ nitrate uptake (Touchette, 1999, supporting an earlier report by Roth and Pregnall, 1988). Moreover, they had elevated total N and amino acid content but negligible accumulation of nitrate, nitrite or ammonium, relative to control plants without water-column nitrate

2

enrichment (ambient nitrate at ,2mM NO ; Touchette, 1999). The data indicate that3

the absorbed nitrate was reduced and assimilated quickly, with limited accumulation of potentially toxic levels of ammonium or nitrite. Instead, an indirect effect of nitrate-induced internal carbon limitation was the apparent underlying mechanism (as previous-ly shown for algae and other plants under high nitrate enrichment; physiological mechanism in Turpin, 1991; Turpin et al., 1991). Significant declines in carbohydrate levels and higher dark respiration were measured in the water-column nitrate-enriched

plants, indicating that the energy- and carbon-demanding process of nitrate uptake and assimilation to amino acids had led to an internal ‘carbon drain’ (Burkholder et al., 1992, 1994; Touchette, 1999).

Zostera marina appears to be especially sensitive to inhibition by water-column

nitrate enrichment. The lack of a ‘shut-off’ mechanism for water-column nitrate uptake may have evolved—as for seagrasses that show lack of product feedback inhibition in sustained ammonium uptake—as a mechanism to enhance survival in oligotrophic, N-limited habitats (Burkholder et al., 1994). In contrast, a physiological inhibitory effect of water-column nitrate enrichment has not been documented for seagrass species

Halodule wrightii, Ruppia maritima, and Thalassia testudinum, using a comparable range of substrate enrichment levels (Burkholder et al., 1994; Lee and Dunton, 1999). Instead, shoot production in these plants was mildly to strongly stimulated by similar enrichment levels.

Nonetheless, unusual nitrate responses among seagrass species are not restricted to Z.

marina. Laboratory cultures of the subtropical seagrass, Halophila decipiens, turned brown and died within a few weeks when nitrate was the only source of N , whereasi

active growth occurred with glutamic acid or ammonium as the N source (Bird et al., 1998). As mentioned, Halophila stipulacea apparently is unable to use nitrate, as well (Doddema and Howari, 1983). Thus, the physiology of nitrate utilization is unusual for some seagrass species, in comparison to many other vascular plants.

A second phenomenon that has been hypothesized to occur in some seagrass species—namely, ammonia toxicity—is a common response to elevated ammonia for vascular plants. For example, research on the seagrasses Ruppia drepanensis and Z.

marina, the freshwater submersed macrophyte Potamogeton densus, and the emergent freshwater plant Stratiotes aloides has revealed significant depression of growth under ammonium enrichment (Mattes and Kreeb, 1974; as described in Roelofs, 1991;

´

Santamarıa et al., 1994). This response to ammonium enrichment was unrelated to light attenuation from algal overgrowth, and ammonium toxicity was suggested as the

´

underlying mechanism (Santamarıa et al., 1994; van Katwijk et al., 1997). Sediment and water-column ammonium were high relative to levels typically found in seagrass

1

habitats, with die-off occurring at 3–220 mM NH4 in the water column, and at

1

´

500–1600mM NH4 in the sediment pore water (Santamarıa et al., 1994; van Katwijk et al., 1997). Furthermore, Z. marina in sandy sediments was more susceptible to this apparent ammonium toxicity than plants grown in organic sediments. Elevated tempera-tures exacerbated the adverse response of Z. marina in sandy sediments to ammonium enrichment (van Katwijk et al., 1997)—similar to the conditions that had exacerbated the inhibitory effect of water-column nitrate enrichment in studies of Z. marina by Burkholder et al. (1992, 1994) and Touchette (1999).

4. Phosphorus physiology

4.1. P acquisition by above- and belowground tissues

Lyngby, 1985). Rhizomes can also absorb P , but uptake rates are approximately 10-foldi

lower than those of roots (Brix and Lyngby, 1985). The importance of above- and belowground tissues in P uptake can vary considerably, with the relative P availability in water-column versus sediment sources as a critical determining factor. In Ruppia

maritima, for example, P uptake by roots was not affected by P availability to leaf tissue,

whereas P availability to roots strongly influenced leaf uptake (Thursby and Harlin, 1984). For plants growing in P-rich waters or sandy (P-poor) sediments, P uptake can be higher in leaves than in roots (McRoy and Barsdate, 1970; Thursby and Harlin, 1984). It has been suggested that Zostera marina assimilates most of its required P for growth through its leaves, and relies upon P uptake from the sediment only when P is negligible in the water column (Brix and Lyngby, 1985).

Factors such as environmental P levels, light, temperature, and tissue age cani

influence the relative importance of P absorption by above- versus belowground tissuesi

(McRoy and Barsdate, 1970; Patriquin, 1972; Penhale and Thayer, 1980; Touchette and Burkholder, 1999). The rate of P uptake is strongly influenced by P availability. For example, P assimilation rates in Z. marina increase with increasing P in the wateri i

column and the sediment pore water (Penhale and Thayer, 1980; Pellikaan and Nienhuis, 1988). As expected, P uptake by belowground tissues of this seagrass was unaffected byi

light availability, whereas leaf P uptake significantly decreased during dark periodsi

(McRoy and Barsdate, 1970; McRoy et al., 1972; Brix and Lyngby, 1985). Similarly, ´

Perez et al. (1994) showed that available light did not affect root P uptake in Zosterai

noltii, but did influence P transport to the leaves. However, P uptake by belowgroundi

tissues of Thalassia testudinum was highest during photosynthetic periods (Patriquin, 1972).

Varying proportions of the absorbed P can be translocated to other tissues. In onei

study of north temperate Z. marina, about 4% of the assimilated P from either leaves or roots was translocated to other tissues within 5 days (usually to younger, actively growing regions; Brix and Lyngby, 1985, in Denmark). Rates of both acropetal and basipetal translocation were depressed during dark periods, and there was no observable P excretion / leakage from above- or belowground tissues. In other research on Z. marina from a warmer temperature regime, Penhale and Thayer (1980) reported that from 1 to

32 23

22% of the assimilated P (tracked by the radiolabeled tracer, [ P]PO4 ) was translocated from roots to other tissues within 12 h; and low losses (3% of the assimilated P) by excretion / leakage were detected from leaves following root uptake. In contrast to the relatively low levels of P release that were observed in that study, McRoy and Barsdate (1970) reported that more than 65% of the P absorbed by belowgroundi

22 21

tissues was translocated to leaves (rates .60 mg P m day ), followed by release to the water within 50 h.

The authors hypothesized that the apparent ‘P pump’ could represent an additional pathway in the P cycle that would allow buried [sediment] P, in both organic and inorganic forms, to re-enter the water column where it would be available to other organisms (McRoy et al., 1972). However, more recent investigations of P excretion in seagrasses indicate that P release from Z. marina and Z. noltii are strongly controlled by

´

the relative P availability in the sediments versus the water column (Perez and Romero, 1993). The quantity of P released was evaluated as insignificant from an ecosystem

21 21

perspective (rates from 10 to 30 nmol P g h , comprising 4% or less of the total

´ ´

assimilated P; Pellikaan and Nienhuis, 1988; Perez-Llorens and Niell, 1993), although such rates could be significant in augmenting the P supplies of epiphytic community that colonized the seagrass plants (Penhale and Thayer, 1980; also see Moeller et al., 1988).

4.2. Phosphorus uptake kinetics

On the basis of sparse reports, P uptake affinities in seagrasses (i a, range 0.12–1.10) are much lower than values reported for active ammonium uptake, but comparable to values for nitrate uptake by leaf tissues (Table 3). From laboratory incubations of

Ruppia maritima and Thalassia hemprichii, Smin for P was lower for roots than fori

leaves, indicating that the roots could initiate P uptake at lower substrate concentrations (Thursby and Harlin, 1984; Stapel et al., 1996). The higher Smin in leaves may limit the period during which leaves can actively absorb P from the water column, thus minimizing the aboveground tissue contribution to the total P budget for these plants (Stapel et al., 1996). In contrast, in eutrophic environments with relatively high P levels

23

(e.g., mean water-column PO4 P at ca. 3.5 mM), it has been estimated that Z. noltii leaves can absorb 2.5-fold more of the P requirement for optimal plant growth

´ ´

(Perez-Llorens and Niell, 1995). As with P uptake affinities, maximum uptake rates

21 21

(Vmax) for P in seagrasses (0.014–43i mmol g dry weight h ) are much lower than those reported for ammonium, and comparable to rates observed for nitrate (Table 3). In addition, P uptake by leaves tends to be more rapid than uptake by roots, despite higheri

Smin values for leaf tissues (Thursby and Harlin, 1984; Brix and Lyngby, 1985). As for N uptake, some seagrasses do not appear to follow the hyperbolic relationships indicative of Michaelis–Menten kinetics in P uptake. A biphasic V versus S curve was described for Zostera noltii, with the first phase characterized by rapid P uptake (firsti

23 21 21

10–20 min; up to 32 mmol PO4 P g dry weight h ), followed by a substantial

23 21 21

´ decline in uptake over time (typically ,6 mmol PO4 P g dry weight h ;

Perez-´

Llorens and Niell, 1995). The initial phase, sometimes referred to as surge uptake, is believed to represent maximum uptake by the plant, whereas the subsequent decline may indicate product feedback inhibition and / or regulation of phosphate efflux (Harrison et

´ ´

al., 1989; Perez-Llorens and Niell, 1995). The maximum P assimilation rates and thei

‘surge’ duration may depend on the nutritional history, wherein P-starved plants would be expected to have higher uptake rates and more sustained (extended duration) uptake. For example, P uptake kinetics measured over 5 h in Thalassia hemprichii from low-Pi

habitat (water column and sediment pore water P ca. 0.3 and 3.9i mM, respectively) did not indicate a biphasic response (Stapel et al., 1996). Rather, there was a sustained period of uptake that lacked apparent product feedback inhibition over the interval tested.

4.3. Phosphorus assimilation

P is generally believed to be taken up by plants using phosphate transporters (Liu eti

al., 1998; Burleigh and Harrison, 1999; Muchhal-Umesh and Raghothama, 1999). These transporters are membrane-spanning proteins that are highly regulated among plants,

both temporally and spatially depending on the prevailing phosphorus conditions in the plant and its environment (Muchhal-Umesh and Raghothama, 1999). Unlike nitrate, the

23

PO4 ion does not undergo reduction prior to assimilation. It enters metabolic pathways through adenosine, wherein a phosphate ester bond is formed between P and ADP toi

produce ATP; or it bonds with hydroxyl groups of carbon chains to form simple phosphate esters (e.g., sugar phosphates; Taiz and Zeiger, 1991; Marschner, 1995). The primary process of P incorporation, through adenosine, occurs in mitochondria duringi

respiration via oxidative phosphorylation, and in chloroplasts during the light reactions of photosynthesis via photophosphorylation (Taiz and Zeiger, 1991).

Because P is critically important in plant metabolism (Marschner, 1995), adequatei

supplies of P are necessary to ensure optimum metabolic performance. Under low Pi i

conditions and / or increased metabolic P demand, plants generally increase phos-i

phomonoesterase activity (that is, the activities of acid- and alkaline-phosphatases or PAs). Increased PA activities enhance the ability of plants to recycle internal P , and toi

utilize P from environmental sources (Vincent and Crowder, 1995). These enzymes tendi

to be substrate-nonspecific, thereby catalyzing the release of P from a broad range ofi

P-containing compounds. The ecological role of PAs, especially alkaline PA, has been linked to phosphorus deprivation in many plants (McLachlan, 1980; Burkholder and

´

Wetzel, 1990; Perez and Romero, 1993; Lapointe et al., 1994). In contrast, acid PA activities tend to be associated with metabolic and developmental patterns, and thus tend to be less variable (Wynne, 1977; Jansson et al., 1988). Therefore, it has been suggested that acid PAs are continually expressed and may be involved in internal phosphorus metabolism, whereas alkaline PAs, with greater external function, are more responsive to environmental P conditions (Vincent and Crowder, 1995).i

In seagrasses, PA activities can vary depending on P availability, tissue age, andi

presence of epiphytes (Table 4). Mature leaves generally have higher PA activity than younger leaves, but the PA activity of mature leaves is believed to be commonly overestimated because of failure to remove most epiphytes while avoiding damage to the plant tissue. Some researchers have attempted to remove the epiphytic colonizers, which can have high PA activity (e.g., Burkholder and Wetzel, 1990), and have reported a trend for increased PA activity with leaf age up to early senescence (Table 4). Epiphyte accumulations can restrict carbon acquisition by macrophytes from the overlying water (Sand-Jensen, 1977), and are believed to restrict water-column P availability to the hosti

plants (Moeller et al., 1988; Burkholder et al., 1990). Thick epiphytic biofilms can effectively block access to the water-column P source (Burkholder and Wetzel, 1990) and, thus, may promote enhanced PA activities in the underlying mature leaves of the host plants.

In aquatic plants under P limitation, PAs may be released extracellularly into thei

surrounding environment (Healey and Hendzel, 1979; Smith and Kalff, 1981; Lapointe et al., 1994), and / or may accumulate internally in both apoplastic and symplastic tissues (Jansson et al., 1988; Thaker et al., 1996). Alkaline PA activity has been inversely related to P availability in seagrasses as well as many other aquatic plants. For example,i

32

declines in alkaline PA activity were observed following PO4 additions in Posidonia

oceanica, and lower PA activities were observed in Cymodocea nodosa from P-rich

´

´

Ferrario-Mery, S., Murchie, E., Hirel, B., Galtier, N., Quick, W.P., Foyer, C.H., 1997. Manipulation of the pathways of sucrose biosynthesis and nitrogen assimilation in transformed plants to improved photo-synthesis and productivity. In: Foyer, C.H., Quick, W.P. (Eds.), A Molecular Approach To Primary Metabolism in Higher Plants. Taylor & Francis, London, pp. 125–153.

Fourqurean, J.W., Moore, T.O., Fry, B., Hollibaugh, J.T., 1997. Spatial and temporal variation in C:N:P ratios,

15 13

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